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Structural and Functional Studies of Polyketide Synthases

Drulyte, Ieva (2018) Structural and Functional Studies of Polyketide Synthases. PhD thesis, University of Leeds.

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Drulyte_I_SMCB_PhD_2018.pdf - Final eThesis - complete (pdf)
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Polyketides, natural products produced by multi-domain polyketide synthases (PKSs), have proven to be excellent starting points for drug discovery. Rational engineering of PKSs holds much promise for the generation of novel polyketide pharmaceuticals, however to enable this we need to gain a better understanding of how the mature polyketides are generated and how individual modules within a polyketide synthase assemble and interact. Here, work was performed to investigate three polypeptides from natural product indanomycin and rhizoxin biosynthesis, including the candidate polyketide cyclase IdmH, the fourth subunit of the indanomycin megasynthase, IdmO, and the branching module from the rhizoxin PKS. Indanomycin needs to undergo several transformations by post-PKS tailoring enzymes. One such enzyme, IdmH, has been hypothesised to act as a cyclase and catalyse the formation of the indane ring via a Diels-Alder reaction. Crystal structure of the wild-type IdmH was determined to 2.7 Å resolution and the interactions between IdmH and its proposed product indanomycin were characterised using NMR spectroscopy and in silico methods. Fully-reducing IdmO module was successfully expressed and purified. Characterisation by negative-stain electron microscopy resulted in a low-resolution model of IdmO, while attempts to carry out cryo-electron microscopy (cryo-EM) analysis revealed a number of difficulties associated with the denaturation of this large complex during cryo-EM grid preparation. A similar cryo-EM approach was utilised to study the branching module from the rhizoxin PKS. A 3.7 Å resolution map was determined for this module containing the ketosynthase, branching and acyl carrier protein (ACP) domains. Two ACP binding sites were identified, which can help explain the unorthodox activity of this module. This research has provided valuable insights into different aspects of PKS biology ranging from polyketide tailoring and branching to the assembly of the intact modules and forms a solid basis for future studies of these fascinating biosynthetic machines.

Item Type: Thesis (PhD)
Keywords: Polyketide synthase, PKS, natural products, indanomycin, rhizoxin, biosynthesis, polyketide cyclase, enzymology, structural biology, X-ray crystallography, NMR spectroscopy, electron microscopy, cryo-EM
Academic Units: The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Dr Ieva Drulyte
Date Deposited: 28 Jan 2019 10:19
Last Modified: 28 Jan 2019 10:19
URI: http://etheses.whiterose.ac.uk/id/eprint/22568

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